Estimation of antimicrobial resistance of Mycoplasma genitalium, Belgium, 2022.

BackgroundAntimicrobial resistance (AMR) of Mycoplasma genitalium (MG) is a growing concern worldwide and surveillance is needed. In Belgium, samples are sent to the National Reference Centre of Sexually Transmitted Infections (NRC-STI) on a voluntary basis and representative or robust national AMR data are lacking.AimWe aimed to estimate the occurrence of resistant MG in Belgium.MethodsBetween July and November 2022, frozen remnants of MG-positive samples from 21 Belgian laboratories were analysed at the NRC-STI. Macrolide and fluoroquinolone resistance-associated mutations (RAMs) were assessed using Sanger sequencing of the 23SrRNA and parC gene. Differences in resistance patterns were correlated with surveillance methodology, socio-demographic and behavioural variables via Fisher's exact test and logistic regression analysis.ResultsOf the 244 MG-positive samples received, 232 could be sequenced for macrolide and fluoroquinolone RAMs. Over half of the sequenced samples (55.2%) were resistant to macrolides. All sequenced samples from men who have sex with men (MSM) (24/24) were macrolide-resistant. Fluoroquinolone RAMs were found in 25.9% of the samples and occurrence did not differ between socio-demographic and sexual behaviour characteristics.ConclusionAlthough limited in sample size, our data suggest no additional benefit of testing MG retrieved from MSM for macrolide resistance in Belgium, when making treatment decisions. The lower occurrence of macrolide resistance in other population groups, combined with emergence of fluoroquinolone RAMs support macrolide-resistance testing in these groups. Continued surveillance of resistance in MG in different population groups will be crucial to confirm our findings and to guide national testing and treatment strategies.

Nationwide Harmonization Effort for Semi-Quantitative Reporting of SARS-CoV-2 PCR Test Results in Belgium.

From early 2020, a high demand for SARS-CoV-2 tests was driven by several testing indications, including asymptomatic cases, resulting in the massive roll-out of PCR assays to combat the pandemic. Considering the dynamic of viral shedding during the course of infection, the demand to report cycle threshold (Ct) values rapidly emerged. As Ct values can be affected by a number of factors, we considered that harmonization of semi-quantitative PCR results across laboratories would avoid potential divergent interpretations, particularly in the absence of clinical or serological information. A proposal to harmonize reporting of test results was drafted by the National Reference Centre (NRC) UZ/KU Leuven, distinguishing four categories of positivity based on RNA copies/mL. Pre-quantified control material was shipped to 124 laboratories with instructions to setup a standard curve to define thresholds per assay. For each assay, the mean Ct value and corresponding standard deviation was calculated per target gene, for the three concentrations (107, 105 and 103 copies/mL) that determine the classification. The results of 17 assays are summarized. This harmonization effort allowed to ensure that all Belgian laboratories would report positive PCR results in the same semi-quantitative manner to clinicians and to the national database which feeds contact tracing interventions.

Performance of the new ID-fungi plate using two types of reference libraries (Bruker and MSI) to identify fungi with the Bruker MALDI Biotyper.

During the last decade, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has revolutionized the diagnosis of fungal infections. Recently, a new Conidia ID-fungi plate (IDFP) medium was introduced to facilitate growth and sampling of fungi. This study aimed to evaluate the IDFP for fungal MALDI-TOF MS identification by comparison with a standard fungal growth medium using two reference libraries. A total of 75 filamentous fungal isolates (including 32 dermatophytes) were inoculated on IDFP and Sabouraud-gentamicin-chloramphenicol (SGC) agar and identified by MALDI-TOF MS using formic acid/acetonitrile extraction. Both the commercially available Bruker library (version 2.0) and the public available MSI web application (version 2018) were applied. For 15% of the isolates, a faster growth was noticed on IDFP compared to SGC. IDFP enhanced the performance of fungal identification compared to SGC for both MSI (increase of 16% identifications to genus and 5% to species level) and Bruker library (increase of 22% identifications to genus and 8% to species level). In total, only 73% of the tested isolates were present in the Bruker library compared to 92% for MSI library. No significant difference (P = 0.46) in MALDI score between IDFP and SGC was observed for the MSI library, but scores were significantly (P = 0.03) higher for IDFP when using Bruker library, potentially explained by the prevention of agar contamination by using IDFP since the Bruker database was created from liquid media. IDFP is a promising alternative growth medium for MALDI-TOF MS fungal identification which would strongly benefit from optimizing the Bruker reference library.

A plethora of manifestations following a Mycoplasma pneumoniae infection: a case report.

Mycoplasma pneumoniae infection can present with a plethora of symptoms and result in a systemic vasculitis by activating a cascade of autoimmune reactions. In this case report, a young man without relevant past medical history was admitted to the hospital with diarrhea, abdominal pain and spiking fever. A CT-scan showed terminal ileitis. A 5-day broad spectrum antibiotic treatment (ciprofloxacin/clindamycin) did not result in any clinical improvement. On the contrary, the patient developed a cholestatic hepatitis, bilateral anterior uveitis and a dry cough. Extensive serological testing finally led to the diagnosis of a M. pneumoniae infection by paired serology (≥4-fold rise in IgG titer). In the diagnostic work-up, a PET-CT was performed and showed increased tracer uptake in the carotids and vertebral arteries, suggesting the diagnosis of vasculitis. After start of azithromycin and low-dose corticosteroids (0.5 mg/kg/day), a gradual clinical and biochemical improvement was observed. But subsequently, the patients relapsed and presented with an acute coronary syndrome. Coronary angiography revealed aneurysmatic deformation of the three coronary arteries, leading to the assumption of coronary vasculitis. Clinical improvement was achieved with high-dose corticosteroids (1 mg/kg/day). This case shows that M. pneumoniae is not merely a pulmonary infection, but that its primary symptoms can be diverse and misleading. All clinicians should be aware of its extrapulmonary manifestations.

Alfa-1-antitrypsin deficiency: a predisposing factor leading to invasive infections?

This case report highlights for the first time a possible link between the presence of alfa-1-antitrypsin deficiency (AATD) and the susceptibility to invasive infections. The current patient, with known AATD, initially presented with nausea, vomiting and headache secondary to Listeria monocytogenes rhombencephalitis. Further on, he developed respiratory insufficiency due to probable invasive pulmonary aspergillosis. Diagnostic work-up could not show any arguments for an underlying immunodeficiency or malignancy. The consecutive course of two rare invasive infections in a healthy individual posed the hypothesis if the underlying AATD could be considered a possible trigger for infections. Indirect clinical observations in literature indeed support this link and in addition, two possible pathophysiological pathways might explain the higher susceptibility for infections in AATD patients. First, alveolar macrophages are dysfunctional in AATD patients leading to a lower apoptotic clearance of bacteria and other (mostly intracellular) pathogens. Secondly, a lower release and lower function of tumour necrosis factor α (TNFα) is seen in alfa-1-antitrypsin depletion, priming the path to more frequent infections, a mechanism that is similar in anti-TNFα treated patients. This case is the first to report on severe or invasive infections related to AATD in humans.

Leptrotrichia Amnionii, an emerging pathogen of postpartum endometritis.

Leptotrichia amnionii, a recently described fastidious gram-negative anaerobic bacterium, is an opportunistic pathogen of the female urogenital tract. We report a rare case of L. amnionii bacteremia in a patient with postpartum endometritis which was successfully treated by amoxicilline-clavunalate. There is more and more evidence that L. amnonii has its role in Pelvic Inflammatory Disease and postpartum endometritis.

Strong and persistent CD4+ T-cell response in healthy adults immunized with a candidate HIV-1 vaccine containing gp120, Nef and Tat antigens formulated in three Adjuvant Systems.

This randomized double-blind study aimed to determine the safety and immunogenicity of a gp120/NefTat candidate human immunodeficiency virus type 1 (HIV-1) vaccine formulated with one of three different Adjuvant Systems (AS02(A), AS02(V) and AS01(B)) in healthy HIV-seronegative adults. All vaccine formulations induced strong HIV-specific CD4(+) T-cell responses characterized by high lymphoproliferative capacity and IL-2 production that were still detectable 18 months after last immunization, with strongest responses seen in the AS01(B) group. Broad coverage was demonstrated against gp120, and to a lesser extent Nef, derived from the most common circulating clades (B, C and circulating recombinant form [CRF]-01). All vaccine formulations exhibited acceptable safety and reactogenicity profiles. The demonstration of superior CD4(+) T-cell induction by AS01(B) provides important guidance for future HIV vaccine development.

Inhibition of replication of primary HIV-1 isolates in huPBL-NOD/Scid mice by antibodies from HIV-1 infected patients.

Although a limited number of HIV-infected patients have broadly neutralizing antibodies, it has not been examined whether these antibodies can protect against infection with primary virus in vivo. Here we screened the plasma of 23 HIV-1-infected patients for broadly neutralizing antibodies. Purified antibodies from subjects with broad and more narrow responses were administered to huPBL-NOD/Scid mice that were subsequently challenged with primary viruses of clade A, B and CRF01_AE. Although we observed a lack of correlation between the data from the in vitro neutralization assay and the results from the passive immunization experiments, we report for the first time that antibodies from HIV-infected persons can inhibit replication of primary virus isolates in an animal model.

The intraspleen huPBL NOD/SCID model to study the human HIV-specific antibody response selected in the course of natural infection.

The intrasplenic injection of human peripheral blood mononuclear cells (PBMCs) into severely immune deficient NOD/SCID mice, causes a massive and transient dominant expansion of human B cells in the spleen. This permits the easy isolation of human monoclonal antibodies specific for different antigens by a Kohler and Milstein-based method. Here we studied the human HIV-specific antibody response in the circulation of mice after intrasplenic transfer of PBMC from untreated HIV-infected patients with detectable to high viral load as well as from HAART-treated and from untreated patients, who kept an undetectable viral load (the latter referred to as "natural suppressors"). Excellent B cell expansion was obtained for all PBMC. High level replication of virus was observed after transfer of PBMC of untreated viremic patients only. A strong and multispecific HIV-specific antibody response was observed after transfer of PBMC of untreated viremic patients and natural suppressors. In contrast, only a weak and pauci-specific antibody response was detected in mice reconstituted with PBMC from successfully treated patients. Based on these observations we conclude that the use of the intraspleen mouse model confirmed a) the presence of HIV-specific circulating memory B cells in untreated patients and natural suppressors; b) the nearly complete absence of circulating memory B cells in patients receiving highly active antiretroviral therapy. Using the intraspleen model we generated large numbers of immortalized B cells and isolated two anti-p24 human monoclonal antibodies. We further conclude that the intraspleen huPBL NOD/SCID model is a small animal model useful for the analysis of the antibody response against HIV found in patients.

Human hepatocytes secrete soluble CD14, a process not directly influenced by HBV and HCV infection.

BACKGROUND: Chronic hepatitis B (HBV) and hepatitis C (HCV) patients have elevated plasma levels of soluble CD14 (sCD14). We examined whether human hepatocytes produce sCD14 in vivo, and whether HBV or HCV infections influence this chimeric production. METHODS: uPA-SCID mice were transplanted with primary human hepatocytes and some animals were subsequently infected with HBV or HCV. Plasma from these mice was analyzed for the presence of human sCD14. The liver was examined via immunohistochemistry. RESULTS: A soluble form of human CD14 could be detected in the plasma from successfully transplanted mice, while it was completely absent in non-transplanted control animals. The isoform of this human sCD14 corresponded with the most abundant isoform found in human plasma. CD14 levels in circulation were not significantly different between non-infected, HBV infected and HCV infected animals. CONCLUSIONS: Our data indicate that human hepatocytes produce sCD14 in vivo, and that liver cells might be the major source of sCD14 in normal human plasma. In addition we demonstrate that HBV and HCV infections have no direct influence on the production of sCD14 by human hepatocytes in this chimeric model.

Soluble CD14 levels are increased and inversely correlated with the levels of hepatitis B surface antigen in chronic hepatitis B patients.

Because it was observed recently that yeast-derived recombinant HBsAg interacts in a lipopolysaccharide binding protein-dependent manner with CD14 expressed on human monocytes, we investigated whether HBsAg influences the serum levels of sCD14, lipopolysaccharide binding protein and C-reactive protein in hepatitis B patients. Samples from acute and chronic hepatitis B and chronic hepatitis C patients were tested. All analytes were measured using commercial assays. HBsAg was quantified using an NIBSC titrated standard. sCD14 levels were higher in chronic hepatitis B and C patients than in healthy controls (P = 0.0006 and P < 0.0001, respectively). In chronic hepatitis B patients an inverse correlation was found between sCD14 and HBsAg (P = 0.0309). Lipopolysaccharide binding protein and C-reactive protein levels were higher in acute hepatitis B patients than in control subjects (P = 0.0217 and P = 0.0034, respectively). In chronic hepatitis B and C, sCD14 and C-reactive protein levels were higher in cirrhotic than in non-cirrhotic patients (P = 0.0072 and P = 0.0223, respectively).

Distribution of human papillomavirus in a family planning population in nairobi, kenya.

BACKGROUND: In sub-Saharan Africa, cervical cancer is the leading cancer among women. The causative role of different human papillomavirus (HPV) types in cervical cancer is established, but the distribution of HPV types within this region is largely unknown. GOAL: The goal was to study the distribution of HPV among family planning clinic attendees in Nairobi, Kenya. STUDY DESIGN: This was a cross-sectional study of persons attending a family planning center in Nairobi, Kenya. RESULTS: HPV data of 429 women were analyzed; 7.0% had low-grade intraepithelial lesions, 6.8% had high-grade intraepithelial lesions, and 0.23% had invasive cancer. One hundred ninety samples (44.3%) were HPV-positive (28.4% were positive for multiple types). The most common HPV types were HPV 52 (17.9% of positive samples), HPV 16 (14.7%), HPV 35 (11.6%), and HPV 66 (9.0%). The risk of high-grade squamous intraepithelial lesions (HSIL) was 88.5 times higher (95% CI, 8.5-1.4 x 10 ) in HPV 16-positive women than in HPV-negative women. Relative risks were 54.3 (95% CI, 4.0-1.4 x 10 ) for HPV 35, 49.2 (95% CI, 3.6-9.5 x 10 ) for HPV 52, and 21.7 (95% CI, 0.0-1.9 x 10 ) for HPV 18. The prevalence of HSIL was not increased in association with HIV-positivity, yet HIV-1 was significantly associated with high-risk HPV types ( P< 0.00001). CONCLUSION: The pattern of HPV distribution in this population was different from that in other regions in the world, which has important consequences for HPV vaccine development.